Nanoscale electronic detection system and methods for their manufacture

ABSTRACT

A new, extremely sensitive, and rapid electronic detection method for direct detection of hybridized genomic targets to specific probes on the microarray is proposed. The method consists of fast electronic accumulation of the DNA target on a particular electrode site at the micro-electrode array, sequential electronic hybridization of oligonucleotide labeled metallic (nano)particles on the target DNA and monitoring the electrochemical AC impedance changes at the electrode site. The method is enhanced by electroplating over the DNA target which serves as the metallization template and over the particles which provide seeds for rapid electroplating. The AC impedance changes are monitored during the electroplating over the DNA target and between the array electrodes sites. The signal in the absence and presence of the target DNA is a difference between “no connection” and a “short” between the array electrodes

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/575,445, filed May 28, 2004, entitled “Nanoscale ElectronicDetection System”, and is incorporated herein by reference as if fullyset forth herein.

FIELD OF THE INVENTION

This invention relates to microscale and nanoscale electronic systemsand methods for their manufacture. More particularly, the apparatus andmethods relate to detectors, especially single sequence detectionsystems.

BACKGROUND OF THE INVENTION

Sequencing of the Human Genome induced a new knowledge in understandingthe correlations between the DNA structure, gene functions, efficiencyof targeted therapeutics as well as occurrence and development of avariety of genetic and infectious diseases. Molecular diagnostics basedon DNA revealed mechanisms and advancement of numerous dangerousdiseases including cancer, HIV, cystic fibrosis, heart and lungdiseases, emerging infectious diseases, to name a few. Although thereare several examples where rapid and sensitive DNA analysis is needed,e.g., infectious diseases and biological agent detection, faster andmore sensitive DNA detection methods will benefit all areas of humanhealth. Recent threats of bioterrorism attacks as well as the appearanceof emerging infectious diseases prompted urgent development of new moresensitive, simple and rapid point-of-use or point-of-care equipment forthe detection and identification of pathogens in various medical orenvironmental samples. Currently, no platforms exist for differentiationbetween common pulmonary infections and emerging infectious pathogens tooffer rapid screening in emergency rooms or in doctor's offices. The DNAmicroarray platform allows a highly multiplexed recognition of a largenumber of characteristic genes.

However, the sensitivity is often not satisfactory for detecting a smallnumber of pathogens or cancer cells in a limited sample volume. The DNAbased technology still relies on the PCR, polymerase chain reactionamplification or similar molecular amplification techniques to enhancethe concentration of DNA. These techniques are time consuming, usuallyrequiring from 30 minutes to 2 hours to achieve a satisfactoryamplification. There is an urgent need for new DNA technologies thatwill require no lengthy molecular amplifications and will be capable ofdirectly detecting specific DNA sequences. Natural DNA hybridizationprocess offers such specificity; however, most standard DNA microarraytechnologies rely on passive diffusion and hybridization of the targetDNA to the probes on the microarray chip which usually takes severalhours to accomplish. Electronically-driven microarray technology (SeeReference(s) 1-8) (e.g., www.nanogen.com, provides fast transport of DNAsequences (less than one minute) to a specific location on the chip. Thedetection is accomplished with fluorophore reporters and laser basedfluorescence detection (See Reference(s) 9-14). The technology utilizesPCR or strand displacement (SDA) amplified DNA as the target samplewhich consumes time and renders these methods incompatible for use inemergent situations where the desirable total analysis time is less than20 minutes.

DNA microarray technology has critical advantages compared to othermethods for DNA based analysis of single nucleotide polymorphisms(SNPs), short tandem repeats (STRs) for human identification or for thedetection of viruses and pathogens because of its inherent possibilitiesfor multiplexed detection on large number of array spots. Recently, anumber of methods for the detection of pathogens or viruses have beendeveloped (See Reference(s) 15-19), however major disadvantages fortheir use as practical portable systems in the field are that they donot satisfy the requirement of highly multiplexed detection. Oftendetection limits, weight limits or accuracy, or the need for skilledpersonnel to operate the instrument, renders those instruments to benon-compliant with the desirable specifications. A portable hospital orclinical lab instrument for DNA based molecular diagnostics should belight-weight (less than around 20-30 lbs), capable of specific detectionof series of genes, characteristic for particular SNPs or pathogens(panels with 20 up to 100 characteristic genes are desirable) withanalysis time less than 0.5 to one hour and detection limits approachingonly few copies of DNA or 10-100 cfu/ml.

In the last decade, the development of microarrays has greatly expandedour analytical capabilities for protein and DNA analysis (SeeReference(s) 20). Many novel techniques now allow us to simultaneouslyanalyze thousands of DNA sequences in microliter volumes at thepicomolar level of sensitivity. Examples include Affymetrix's GeneChip™(See Reference(s) 21-23), Nanosphere's (See Reference(s) 24) goldnanoparticle technology and Nanogen's electronically active Nanochip®(See Reference(s) 25) technologies. Assays have been developed for geneexpression analysis (See Reference(s) 26), forensics (See Reference(s)27), SNP (See Reference(s) 28) analysis and a host of other novel assayformats.

Competitive DNA based portable systems are developed today mostly forthe emergent applications such as the detection of biological warfareagents or pathogens. These include Idaho Technology's RuggedizedAdvanced Pathogen Identification Device (R.A.P.I.D System) and RapidCycler systems (See Reference(s) 29), Autonomous Pathogen DetectionSystem (APDS) developed at LLNL (See Reference(s) 30-31), and Cepheid'sSmart Cycler system (See Reference(s) 32-34) that are capable ofintegrating on-chip lysis of microorganisms, amplification of theircharacteristic DNA through the polymerase chain reaction andfluorescence detection of DNA. Although many of these systems offerelegant solutions to detection of a smaller number of agents, the numberof optical channels installed limits their application when a largernumber of agents or genes needs to be detected. Compared to thosetechnologies, the microarray platform practically does not pose a limitto multiplexed detection of large number of pathogens as well as theircharacterization by multiple genes. Today no portable point-of-caremicroarray based DNA analysis system has been developed for commercialuse.

Recently, the Nanochip® microarray technology has developed a portableelectronic microarray system which accommodates an electrode array with400 sites and uses fluorescence based detection for the detection ofaddressed DNA targets. Assays for Factor II and V, SNPs for humanidentification based on mitochondrial DNA, as well as assays foremerging infectious disease and biological warfare pathogens have beendeveloped.

All of the above techniques utilize PCR or similar molecularamplification techniques to amplify the DNA target in the sample. Thisproposal initiates the development of a new direct electronic DNAdetection technique which will not need PCR or other long-termamplification methods to amplify the DNA concentration in the sample.The method will provide a new microarray-based platform for extremelyrapid DNA analysis which will be highly sensitive and specific for aparticular set of targeted genes. The electronics-based detectiontechnique will allow design of a small, portable, potentially hand-heldmicroarray instrument and will not need more complex and field-sensitiveoptical detection system consisting of sensitive lasers, lenses andother optical components.

The intrinsic conductivity of bare DNA is too low to allow itsutilization as a molecular wire or to directly measure its presencethrough simple conductance measurements between two electrode sensors(See Reference(s) 35-36). The localization and binding of few target DNAmolecules between the electrodes or on the substrate at a desiredlocation is extremely slow because this step is controlled by a slowdifflusion process. If the concentration of the analyte is only a fewmolecules of DNA the passive process of capturing DNA has very lowstatistic probability. The proposed technology easily overcomes theseproblems by directional and fast electrophoretic transport of DNAtargets toward the electrode array sites.

Several different DNA metallization techniques have been reported (SeeReference(s) 37) utilizing various metals, including silver (SeeReference(s) 38), palladium (See Reference(s) 39), and platinum (SeeReference(s) 40). In general, those methods are based on electro-lessplating processes which usually consist of two steps. Metallic clustersare first formed on the DNA, and then used as nucleation sites forselective metal deposition in a subsequent metal reduction process untila continuous metallization of the DNA molecule is obtained. Theformation of metallic nucleation centers relies on binding of metal ionsor complexes to the DNA and their subsequent reduction to form metallicclusters, or on binding of small metallic particles to the DNA.

These metallization techniques suffer from several drawbacks. First,these metallization processes are very slow, particularly if based onparticle binding to DNA. They are uniform over the entire DNA scaffold,thus non-specific as well as yield to a highly non-specific depositionof metallic ions or metallic particles on the substrate at locationswhere no DNA is present causing a high level of false positive signals.More importantly, electro-less metallization processes destroy therecognition properties of the DNA, thus preventing any subsequentreporter binding steps through hybridization. A molecularlithography-based method has been recently developed which provides somelevel of protecting specific sequences of the DNA molecules from themetallization process (See Reference(s) 41). The method involves themetallization of DNA molecules by sequence-specific derivatization withglutaraldehyde, which acts as the localized reducing agent on the DNA.Silver ions are then specifically reduced by the DNA-bound aldehydegroups in the aldehyde-derivatized regions, resulting in the formationof a silver cluster chain along the DNA. An electroless gold depositionprocess (See Reference(s) 42), catalyzed by the silver clusters is thenused to generate continuous DNA-templated gold metallization. Theprocess consists of a number of cumbersome steps which require severalreagents that need to be freshly prepared.

A recent review article by J. Wang (See Reference(s) 43) summarized thedetection techniques for DNA templated metallization. His group hasdeveloped an electrochemical based technique in which deposited silverions are reduced and subsequently dissolved. The silver ionconcentration is then determined using anodic stripping voltammetry(ASV). This technique although highly sensitive for determination ofsilver ions is prone to high false positive results, because a singlesilver particle adsorbed at the substrate and not on the DNA moleculewill produce a high silver ion ASV signal. Mirkin's group is one of thegroups leading the innovation in applying nanoparticle-DNA assemblies tonanofabrication and sensor applications (See Reference(s) 44-47). Theyhave developed an electrical DNA detection method utilizingoligonucleotide ftinctionalized gold nanoparticles and closely-spacedinterdigitated microelectrodes (See Reference(s) 48). Theoligonucleotide probe was immobilized in the gap between the twomicroelectrodes. The gold nanoparticles are attached to the DNA targetover the oligonucleotide probes. The method involves a subsequent silverdeposition which leads to a measurable conductivity signal. The methodshowed a high sensitivity with a 0.5 pM detection limit. The methodproposed in this project differs from this technique in directed andcontrolled electrophoretic accumulation of both DNA target andoligonucleotide labeled metallic particles as well as introduceselectrophoretic amplification of the signal by clustering metallicparticles on the template DNA. This assures a high signal-to-noise ACimpedance signal measurements of the metallic particles clustering onthe metallized DNA through a repeated and/or cyclic electrophoreticprocess where metallic particle tags yield an amplified signal. Theproposed method utilizes a fast directed electroplating of target DNAtemplate as opposed to the sterically non-specific electroless plating.The technical principles of the proposed detection method are summarizedin a separate section below.

Nanogen's microarray technology (http://www.nanogen.com) is unique amongDNA microarrays due to the use of electrophoretically driven, activetransport of the DNA analyte and/or probe molecules at the array. Thetransport over the array is electronically controlled by connecting thearray sites as electrodes. This electronic addressing of biomolecules atthe array can accelerate molecular binding on the microchip up to 1,000times compared to the traditional passive methods. For instance,hybridization on passive microarrays may take up to several hours whichis critical when low concentrations of DNA target need to be determined.The most recent version of the Nanochip® is an array of 400 platinumelectrodes, 50 pm in diameter, each of which is independently controlledand monitored by circuitry designed into the chip. A thin, hydrogelpermeation layer containing co-polymerized streptavidin, covers thesurface of the microarray electrodes. The main function of the hydrogelmatrix is to provide binding sites for biotin labeled DNA probes;however, it also protects the DNA from the harsh electrochemicalenvironment at the electrode surface. We have taken advantage of the H⁺generated at the positive electrode to perform electronic hybridizationwhich promotes conditions for efficient DNA hybridization inzwitterionic buffer such as histidine. Nanogen's commercial instruments(Nanochip® System) can use electronic, thermal or chemical techniques,depending on the application, for precise, accurate stringency control.This provides an extremely flexible platform for the assay designallowing several types of multiplexed analyses, e.g. determination ofmultiple genes in one sample, multiple samples with one gene, ormultiple samples with multiple genes. The ability to control individualtest sites permits biochemically unrelated molecules to be usedsimultaneously on the same microchip. In contrast, sites on aconventional DNA array cannot be controlled separately, and all processsteps must be performed on an entire array. The commercial system usesfluorescence based detection using fluorophore labeled oligonucleotideprobes or reporters.

Prior patents relating to the use of microarrays for nanofabricationinclude the following, all of which are hereby incorporated in byreference as if fully set forth herein: U.S. Pat. No. 6,652,808 entitled“Methods for the Electronic Assembly and Fabrication of Devices”, U.S.Pat. No. 6,569,382 entitled “Method for the Electronic, HomogenousAssembly and Fabrication of Devices”, and U.S. Pat. No. 6,706,473entitled “Systems and Devices For Photoelectrophoretic Transport andHybridization of Oligonuceotides”.

SUMMARY OF THE INVENTION

A new, extremely sensitive, and rapid electronic detection method fordirect detection of hybridized genomic targets to specific probes on themicroarray is proposed. The method consists of fast electronicaccumulation of the DNA target on a particular electrode site at themicro-electrode array, sequential electronic hybridization ofoligonucleotide labeled metallic (nano)particles on the target DNA andmonitoring the electrochemical AC impedance changes at the electrodesite. The method is enhanced by electroplating over the DNA target whichserves as the metallization template and over the particles whichprovide seeds for rapid electroplating. The AC impedance changes aremonitored during the electroplating over the DNA target and between thearray electrodes sites. The signal in the absence and presence of thetarget DNA is a difference between “no connection” and a “short” betweenthe array electrodes. This assures an extraordinary signal-to-noiseratio. The method offers unprecedented sensitivity, theoreticallyapproaching single or only a few DNA molecules attached to the electrodesite. Rapid electronic addressing of the DNA target and labelednanoparticles to the microarray assures that the detection at theselevels of sensitivity will be achieved within only a few minutes.

Applications of the innovations used in the electronic detection systeminclude at least the following: electronic capturing of target DNA onthe electroactive microarray and electronic alignment of labeledparticles as seeds for DNA-templated electroplating, sequential orcyclic electrophoretic accumulation of labeled particles on DNA targetas tags for AC impedance signal amplification—cyclic electrophoretic ACsignal amplification, and DNA-templated electroplating on theelectroactive microarray—electroplating of target DNA between theelectroactive array sites over the labeled metallic particles and/ordirectly in the presence of electroplating ions, e.g., Ag, Au, Pd.

Portable DNA analysis systems for molecular diagnostics is theintegration of the sample preparation and detection steps on a singleplatform. This invention includes an electronic detection technique forthe microarray technology which will be capable of easy integration withvarious sample preparation methods including those based on magneticparticles.

The target DNA-templated electroplating detection system which utilizeselectrochemical impedance spectroscopy between the electrode array sitesas the microarray detection signal presents an innovative approach toDNA sensing. However, the detection technique builds on similar,established, and demonstrated electro-less techniques for DNAmetallization which utilize charge interactions between the metallicions and DNA and subsequent reduction of attached metal ions. Other suchtechniques utilize micro- or nanoparticles attachment to the DNAstructure to achieve a layer of metallic particles which are thenpassively coated using a different set of metallic particles. Thesetechniques often take hours to implement the DNA plating process and arenot site specific. The unsurpassable advantage of the proposed detectionsystem is that the DNA target as well as the metallic particle tags arevery rapidly and specifically addressed at the electroactive microarray,they can be easily accumulated at a particular array site and AC signalenhanced in a cyclic electrophoretic accumulation of particle tags. Thisunique and rapid signal enhancement by electrical alignment andelectronic formation of metallic particle clusters on the target DNAassures an easily measurable electrochemical impedance changes on theelectrode site. In addition, the electronically aligned particles enablefast seeding of the DNA template as well as extremely accurate DNAelectroplating. The use of direct and sequence specific electroplatingof DNA, instead of slow electro-less plating techniques, is proposedhere for the first time.

The electroactive transport allows attachment of metallic tags on DNA ina cyclic and amplifiable manner where each cycle occurs within only afew seconds. The basic electronic microarray technology will allowdevelopment and unrestricted practicing of this new “electrophoreticamplification” technique for rapid enhancement of the DNA signal. Asimilar amplification technique may be used in a fluorescence baseddetection where the DNA and fluorophore attached tags are accumulatedand amplified using electroactive transport. This project will focus onAC impedance based detection of the signal that is highly compatiblewith our electronic microarray technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the proposed electronic detectionsystem for specific and highly sensitive detection of DNA targets.

FIG. 2 shows how the AC signal monitoring is performed on themicroarray.

FIG. 3 shows the use of several types of metallic particle tags.

FIG. 4 shows simultaneous detection of all five HPV types.

FIG. 5 demonstrates that it is possible to perform simultaneous on-chipSDA amplification of up to 10 different genes in a single sample.

FIGS. 6 a and 6 b show the AC impedance spectra which demonstrateschanges in capacitive and resistive components occurring between twoelectrode array sites (the locations 1,1 and 1,10 are shown; the firstnumber designates row and the second number designates colurn in themicroarray) at two working electrode potentials applied with respect tothe chip reference electrode and as a function of the histidinesupporting electrolyte concentration.

FIG. 7. Nanogen's portable prototype instrument with the electroactivemicro-array and optical detection. The instrument is operated by alaptop (left). Components of the instrument include the cartridge inletport, reagent reservoirs, peristaltic pumps, electronic control andoptical detection system with a CCD camera (right).

FIG. 8. Photograph of the 400-site CMOS ACV400-chip cartridge and array.Four counter-electrodes, two longitudinally and two horizontallypositioned surround the active working electrode array.

DETAILED DESCRIPTION OF THE INVENTION

AC Impedance System for Detection of DNA-Templated Electroplating

FIG. 1 shows a schematic diagram of the proposed electronic detectionsystem for specific and highly sensitive detection of DNA targets. Thedetection consists of the following steps.

Electronic addressing of the target DNA occurs first. This step occursin accordance to Nanogen's developed technology and implies accumulationof low concentration of DNA targets at an electrode array site fromsolution by electrophoresis. The electronic microarray is covered by ahydrogel permeation layer (ca 7-10 micron thick) containing streptavidinmolecules. The proposed system assumes the use of pre-loadedbiotinilated probes complementary to a particular gene region ofinterest on the target DNA. The target DNA can be very rapidly, withinless than one minute, accumulated from the solution and electronicallyhybridized at a particular array site, providing a localization of thedetection process.

Once the target DNA molecules are hybridized to the oligonucleotideprobes at the electrode, the oligonucleotide labeled metallic particlesare hybridized along the captured target DNA. The sequences of the oligoprobes on the particles will vary depending on the size of the DNAtemplate and the diameter of particles, thus providing a proper spacingbetween the particles on the DNA for subsequent metallization. The useof oligo-labeled particles offers an additional level of specificitythus reducing the non-specific binding and occurrence of eventual falsepositives to a minimum.

The non-captured particles are washed away and those captured on the DNAtarget(s) are electrophoretically aligned, thus providing a series ofseeding sites for continuous metallization.

Nanogen's technology allows precise spatial capturing of DNA targets ata particular microarray site and this feature is used further in theproposed system for localized DNA-templated electroplating. Theelectroplating occurs first through the nanopores (diameter ca 50-200nm) of the thin permeation layer and proceeds to the first and thensubsequent metallic particles aligned (hybridized) along the DNA target.Because the size and number of particles can be optimized with respectto the DNA target, the electroplating process can be accomplished withinonly few minutes.

The accumulation of metallic particles at the captured DNA template canbe followed through changes in the AC impedance signals at the electrodesite. FIG. 2 shows how the AC signal monitoring is performed on themicroarray. The electrochemical double layer is formed on the electrodewhere the DNA target is accumulated and extends through the pores of thehydrogel permeation layer. As the metallic microparticleselectrophoretically accumulate on the DNA target template they screenthe electric field lines extending through the solution between the twoelectrode array sites and particularly change the capacitive and/orresistive components of the impedance of the working electrode (theelectrode where the DNA target is addressed). Each metallic particlepossesses its own electrochemical double layer. The thickness of theelectrochemical double layer (EDL) typically ranges from 10 nm to 100 nm(See Reference(s) 49). Thus each particle can further disturb theimpedance signal of the electrode through its own capacitive componentof the particle EDL. In addition, the aligned metallic particles can actas a series of bi-polar electrodes inserted between the two electrodearray sites.

These processes could accelerate the electroplating process over theseed-particles as well as affect the electrode EDL. It is envisionedthat the AC impedance signal will change significantly as the particlesaccumulate even without the electroplating process. However, for highersignal sensitivity, the aligned metallic particles are electroplated.They become electronically connected to the electrode array site andeffectively extend the working electrode surface area. The use ofnanoparticles will significantly change the electrochemical surface areaof the working electrode and provide an easily measurable AC impedancesignal change. The proposed detection technique envisions even furthersignal amplification which occurs when the metallic particles bridge thegap between the two electrode array sites. The bridging can be promotedby using relatively long oligonucleotide probes at the electrodes aswell as by minimizing the electrode spacing.

FIG. 2 shows an AC impedance monitoring of the target DNA-templatedelectroplating process using electrophoretically accumulated and alignedmetallic particles along the DNA target captured at a particular arraysite. Two cases are shown, one that demonstrates the changes in theelectrode impedance due to clustering of metallic particle tags andtheir effect on the electric field lines (dashed lines) (left) and theother when the metallic particles bridge the gap between the electrodesites (right).

Poly-T Embodiment

There is yet another signal amplification technique that is a part ofthe proposed electronic detection technique and can be used if very lowconcentration of DNA target needs to be detected. FIG. 3 shows the useof several types of metallic particle tags. The first step includeselectrophoretic addressing of metallic particle labeled with botholigonucleotides having a complementary sequence to the target DNA andoligonucleotides having a simple repetitive sequence such as poly-Ttails. Other simple sequences could be used. A second type of metallicparticle tags contains oligonucleotides complementary to the poly T,i.e., a poly A sequence (or similar simple sequence complementary to thesequence on the first set of particle tags). The method implies arepetitive electrophoretic addressing of metallic particle tags which insubsequent addressing steps hybridize between themselves, thus promotinga fast clustering of metallic particles at the electrode site where theDNA target is captured. This will cause dramatic changes in the ACimpedance signal because a large percentage of the electrode area couldbe covered quickly. This new “electrophoretic amplification” of thesignal uses fast electrophoretic addressing of multiple particle tags inseveral separate steps or cycles (a washing step may be needed betweenthe additions of particle tags). Because the second addition or thesecond cycle already provides a chain-like hybridization between theparticle tags, it is envisioned that only few such cycles may be neededto obtain a high signal-to-noise ratio. The electrophoretic addressingin each cycle will take only a few tens of seconds, thus the entirecyclic amplification process will be no longer than 3-5 minutes. Thisnew signal amplification technique can yield to an extremely fast andhighly sensitive DNA detection system.

The cyclic electrophoretic addressing also implies the addressing ofparticle tags of opposite charge. Some metallic particle tags can bemade negatively charged (e.g., carboxylated particles) or positivelycharged (e.g., aminated particles). These particle tags will contain thesame type of oligonucleotide labels as described above. The advantage ofthis approach is that once the DNA template is electronically hybridizedand anchored to the permeation layer, these metallic particles can beaddressed in a faster, electrochemical “stirring” mode by repetitivelyreversing the polarity of the two electrodes (one contains the DNAtarget the other is the counter electrode). The tags in the second orthird cycle could be added simultaneously and the chain-likehybridization and clustering induced by a polarity reversal.

FIG. 3 shows enhancement of the AC impedance signal through cyclicelectrophoretic hybridization of various metallic particle tags capableof a chain-like hybridization between themselves. This can occur in onlya few fast cycles as well as by using the particles of an oppositecharge and by reversing the polarity of the electric field applied atthe electrode site.

Experimental

Nanogen, Inc. has previously designed and developed miniaturized andintegrated systems for microarray-based DNA detection (See Reference(s)50-52). Nanogen's technology for DNA detection (commercial Nanochipgelectronic microarray system) enables rapid and accurate determinationsof single nucleotide polymorphic mutations (See Reference(s) 53).Nanogen offers commercial analyte specific reagents for the diagnosis ofa number of coronary and hemochromatosis diseases (e.g., Factor II,Factor V, Factor V/II combination assay, cystic fibrosis, HFE, Canavandisease and ApoE gene—late onset of Alzheimer's disease). Nanogen'splatform is a unique and open platform which allows customers to createtheir own arrays and assays. Customer list of applications based on SNPdetermination using our platform includes: coronary artery diseases,cardiovascular disease, hypertension, cardiac function, cancerapplications, bacteria identification, multidrug resistance, hemophilia,Thalassemia, etc.

Sensitive Detection of Infectious Disease Pathogens Using ElectronicMicroarray

This section summarizes recent studies performed at Nanogen todemonstrate efficient electronic accumulation of PCR and SDA (stranddisplacement) amplified DNA targets on the electronic microarray and itsdetection using current fluorescence based detection. A feasibilitystudy was performed using five Human Papillomavirus, HPV types (HPV 16,HPV 18, HPV 31, HPV 33 and HPV 45). The amplification was performedusing PCR (AmpliTaq® Gold) in 25 μL reactions on a Perkin-Elmer 9700.Detection was performed on a 100-site chip. FIG. 4 shows simultaneousdetection of all five HPV types. All five types clearly show significantsignal above the background signal. Three out of five of the HPV typeswere present at only 10 copies).

FIG. 4 shows fluorescence data obtained on 100-site electronicmicroarray for detection of five HPV types amplified using PCR.Detection as low as 10 copies of each HPV type was demonstrated.

A multiplexed PCR-based assay for Bacillus anthracis and vaccinia wasdeveloped and an independent validation was performed by ourcollaborator, Midwest Research Institute. Testing included evaluation ofscreening assays and confirmation assays using hemagglutinin gene forvaccinia and CapB and protective PA genes for anthrax. Specificity ofthe assays was evaluated against a panel of 28 anthrax strains and nearneighbors of B. anthracis, vaccinia, rabbitpox, raccoonpox, and a numberof other select agents including Francisella tularensis, Yersiniapestis, Clostridium botulinum, and Erwinia Herbicola. The proceduresincluded: (i) overnight growth of B. anthracis strains (available fromATCC), vaccinia, and all competitive strains used; (ii) extraction oftheir DNA using bead beating, centrifuging and elution in accordance tocommercial kits (modified Qiagen kits); (iii) DNA quantitation(PicoGreen dsDNA Quantitation kit, Molecular Probes), and (iv)performance of: a) screening assay; b) confirmation, competition assays,and c) specificity assays. The experiments were conducted under BSL 3safety conditions when needed. The limits of detection (LOD) weredetermined for the range between 0.17 to 1,700 copies of B. anthracisstrains (per PCR reaction) or 0.0015 to 1,500 PFUs for vaccinia usingserial dilutions of quantified DNA. (50 microliter PCR reactions wereperformed on a PE 9600 thermocycler and detection accomplished onNanogen's 100-site electronic microarray). Testing of B. anthracis(Vollum strain) demonstrated a limit of detection of 1 pg or 170 copiesfor the CapB screen assay (100% positive results for 20 replicates), and10 pg or 1,700 copies for the PA gene. The confirmatory assay for theCapB gene showed LOD of 100 fg or 17 copies (100% positive results for20 replicates). Testing of vaccinia, ATCC VR-2010, with the Hema assaydemonstrated an LOD of 15 PFU (plaque forming units) The specificitytesting (see list of near neighbors and other select agents testedabove) demonstrated that positive results were obtained only when targetgenes CapB, PA, or Hema were present in the sample and no other agentsinhibited the positive results. One ng DNA per reaction (170,000 copies)was used in the specificity testing.

On-chip Strand Displacement Amplification—Demonstration of a HighlyEfficient Accumulation of DNA Targets

We have developed a number of assays using an isothermal StrandDisplacement Amplification (See Reference(s) 54-55). (SDA, licensed fromBectkon Dickinson) of DNA targets because this method requires a muchsimpler device for thermal control in a portable instrument compared tothermal cyclers used for the PCR amplification. In the SDA amplificationDNA polymerase recognizes the nicked strand of DNA and initiatesre-synthesis of that strand, displacing the original strand. Thereleased amplicons then travel in solution to primers for thecomplementary strand which are either in solution or anchored.Oligonucleotide primers without nicking sites called bumper primers aresynthesized in the regions flanking the amplicons just produced, andassist in strand displacement and initial template replication. Atypical reaction mix for SDA amplification consists of the followingmaterials.: a) sense and antisense primers 500 nM; b) Bumper primers 50nM; c) dNTP mix 1.4 mM each; d) Bst polymerase 9.6 U/rxn; e) Bbv nickingenzyme 3.75 U/rxnMg(OAc); f) 10 mM pH 7.6 phosphate buffer, 25 mM.Generally, the reaction volume is 10-50 μl. These parameters areoptimized through the Design of Experiment (DOE) optimization ofexperimental parameters. We have developed anchored SDA amplificationmethod where the internal amplification primers (not the bumper primers)are biotinylated and addressed to specific electronic microarray siteswhere they bind to the streptavidin in the hydrogel permeation layer.These primers can be pre-loaded on the chip at a manufacturing stage.Preliminary stability experiments performed in the period of ca 2 monthsdemonstrated good stability of pre-loaded primers. This step will beimportant in accelerating the assays and performing the addressing oftargets and reporters only. The target DNA is then addressed to thearray site where it electronically hybridizes to the anchored primers.Finally, the microarray is covered with a reaction mixture containingenzymes, bumper primers and dNTP's and heated to 50° C. for 30 minutesto an hour to obtain the reaction products.

FIG. 5 shows a 10-plex on-chip SDA amplification. The pattern ofamplified genes is shown on the left. On the right is a fluorescenceimage of the microarray after amplification and reporting. (NatureBiotechnology, Feb. 2000).

FIG. 5 demonstrates that it is possible to perform simultaneous on-chipSDA amplification of up to 10 different genes in a single sample. Theexperiment shows multiplexing of 5 human and 5 bacterial genes relevantto identification of infectious diseases and/or biological warfare agenton the electronic microarray. A number on-chip SDA based assays weredeveloped for the detection of infectious pathogens and/or biologicalwarfare agents using our miniaturized prototype microarray detectioninstrument (shown in FIG. 7). The following 6 genes for four biologicalwarfare agents were analyzed: bacillus anthracis (anthrax) (cap B and PAgenes), vaccinia (hemagglutinin gene), Staphylococcus aureus (sea andseb genes) and plague (Yersinia pestis) (plasminogen activator, PLAgene). A range of concentrations of each DNA was addressed before theanchored amplification. Concentrations as low as 85 copies ofDNA/microliter (in the detection chamber) could be commonly detected forvaccinia. For the B-list CDC agents, e.g., E. Coli and S. typhymurium,results obtained reproducible anchored SDA data in the range between10-100 copies of DNA (with respect to the starting volume of theamplification reaction). Very recently, we have demonstrated that as lowas 5 copies of the vaccinia DNA target gene per array electrode can beefficiently accumulated within 1 minute of electronic addressing time(results obtained using a portable instrument). Positive results wereobtained on 18 addressed electrodes using 85 copies/microliter on thechip (yielding 85/15 ca 5 copies per electrode). The result was obtainedafter SDA amplification of the DNA concentration on each electrode arraysite. This demonstrated that the electronic addressing is efficientenough to be used in the proposed direct amplification-less detectiontechnique where only a few DNA molecules present in the sample can beefficiently captured on the electrode array site. This resolves one ofthe problems in the proposed detection technique, i.e., a demonstrationthat one or few DNA target molecules can be attached to the array sitewithin a very short period of time (one minute).

AC Impedance Measurements on the Electronic Microarray

We have performed initial AC impedance measurements between theelectrode array sites on the 400-site microarray in conditions whereelectrophoretic DNA accumulation is promoted. The AC impedance spectrashown in FIG. 6 a and 6 b demonstrate changes in capacitive andresistive components occurring between two electrode array sites (thelocations 1,1 and 1,10 are shown; the first number designates row andthe second number designates column in the microarray) at two workingelectrode potentials applied with respect to the chip referenceelectrode and as a function of the histidine supporting electrolyteconcentration. The spectra exhibit typical Randles equivalent circuitcircular shape (cf., FIG. 6). By increasing the concentration ofhistidine the semi-circles become smaller, indicating a higher currentdue to higher concentration of the electroactive species in solution.Polarization resistance, Rp, and solution resistance, R_(s,) werecalculated for all impedance spectra using a least-square method fit fora semicircle as shown in FIG. 6. At low conductivity, i.e., at lowelectrolyte concentrations, the R_(p)/R_(s) ratio at E_(DC)=0.0 V is upto two orders of magnitude higher compared to higher electrolyteconcentrations. This trend is also observable at E_(DC)=1.3 V, althoughthe decrease in the R_(p)/R_(s) ratio with concentration is smaller,i.e., up to 10-fold decrease. The results clearly demonstrate that atlow electrolyte concentrations the total resistance is very high, and isdominated by the polarization resistance. Consequently, the totalcurrents at the electrode array are very small and solution impedancecan be affected by the geometrical arrangement of the electrodes. Theseimpedance characteristics precisely describe relevant conditions of ourassays on the electronic microarray. The data indicate that theelectrode/electrolyte interface on the array sites will be significantlyaffected by the presence of adsorbing species on the electrode as wellas by any changes in the electrode geometry. Accumulation of metallicnanoparticle tags, in particular their exponential amplification in thechain-like electroplating of the DNA target, can dramatically increasethe electrode electrochemical surface area and yield an easilymeasurable impedance signal dependent on the DNA concentrationaccumulated at the array site.

Research Design and Methods

The overall objective of the proposed research is to demonstrate thefeasibility aspects of the development of a new microarray detectionplatform that uses electronic addressing of a low copy number of DNAtargets and its detection using the electrochemical AC impedance signalsof the metallization process of the DNA target molecules attached to thearray sites. Metallic particle tags are used to enhance the AC signalduring the DNA-templated electroplating and the signal is amplifiedthrough a cyclic electrophoretic addressing of the particle tags. Theelectronic detection system enables the cartridge as well as theinstrument packaging in a miniaturized format which will allowdevelopment of a portable instrument. The proposed research willleverage our previous efforts in the development of portable DNAmicroarray instrumentation and an existing prototype miniaturizedplatform containing the necessary fluidics; electronic and softwarecomponents will be adapted and used in the validation of the proposeddetection system. The following are the specific technical objectives ofthe proposed research.

AC impedance detection of the DNA metallization process in the presenceof metallic particle tags.

Design and fabrication of an electronic microarray and cartridge withthe electrode array geometry suitable for the proposed detection system.

Demonstration of amplification-less, rapid and sensitive detection ofDNA target molecules.

Design and testing of a representative assay and validation of thedetection system.

Experiments planned in the proposed research and development effort willbe entirely performed at Nanogen's facilities. The masks for thefabrication of microarray chips will be outsourced to a siliconmicromachining foundry and the fabrication of the array and cartridgewill be made in-house using methods and vendors established for ourcommercial equipment. Nanogen has all the necessary equipment,microfabrication facilities (clean rooms, class 100 and 1000),microbiology and molecular biology labs as well as personnel availableto perform all the tasks of the project.

AC Impedance Detection of the DNA Metallization Process in the Presenceof Metallic Particle Tags

Electrochemical impedance spectroscopy (See Reference(s) 56) utilizes asmall 10-50 mV sinusoidal potential signal applied in a range offrequencies (from few micro-Hertz to MHz range) at the working electrodeto determine the capacitive and resistive components at theelectrode/electrolyte interface. The method allows a mechanistic insightinto the structure of the electrochemical double layer (capacitivebehavior), discriminates faradaic or electoractive components of thecurrent and difflusion controlled processes as well as it providesresistive or capacitive behavior of a coating or adsorption on theelectrodes. The impedance is usually expressed as a complex ftnction(cf., Eq 1-3) and data are represented using Nyquist plots where real orresistive components are presented on the X-axes and imaginary orcapacitive components are represented on the Y-axes (cf., FIG. 6). Bodeplots are used to examine a phase shift and absolute value of impedanceas a function of frequency. An electronic equivalent circuit is usuallyestablished which provides a model of the interface and helps withunderstanding the dominant real time (resistive) or imaginary(capacitive) components of the impedance signal as the experimentalconditions are varied.E(t)−E ₀ exp(jωt)  (1)I(t)−I ₀ exp(jωt−jφ)  (2)

Where E is a sinusoidal potential applied, and I is the currentresponse, ω is angular velocity. The impedance can then be representedas the complex number:Z=E/I=−Z ₀ exp(j, φ)=Z ₀(cos φ+j sin φ)  (3)

AC impedance was recently used to detect an antibody/antigen bindingeffect at the flnctionalized electrodes (See Reference(s) 57). Theseresults showed 25% difference in AC signal comparing electrodesflnctionalized with a specific antibody and another electrode with acontrol (non-binding) antibody. These examples as well as some of ourpreliminary data, demonstrate that the AC impedance studies are suitableto monitor the adsorption or deposition processes occurring directly onthe surface of the electrodes, thus reflecting minimal changes in thesurface area of the working electrode. The particle accumulation,especially when enhanced by cyclic electrophoretic addressing on DNAtarget molecules, will affect the electrochemical double layer extendingthrough the hydrogel layer nanopores and cause substantial impedancechanges during electroplating of DNA targets and metallic particleseeds.

The following is a rationale of the proposed experiments: 1. ACimpedance spectra will be established and compared in the presence andabsence of captured DNA targets on a number of microarray electrodesites and reproducibility of the signal established; 2. Fundamentalchanges of impedance parameters will be investigated for theelectroplating process at a particular electrode site; 3. Impedancesignals will be determined for DNA-templated electroplating process inthe presence and absence of metallic particles addressed at the DNAtarget; 4. Dependence of the most prominent impedance component of theAC signal will be examined as a function of the concentration of DNAadded to the array fluidic chamber. The DNA targets will include severallevels of complexity: a) initial optimizations will be performed withPCR amplified and purified genomic DNA (size in the range 200-1,000 bp)with known sequences (genomic DNA available from ATCC; DNA targets anddesigned primers are available for a number of pathogens, e.g., Yersiniapestis, pla gene, Lysteria monocytogenes, hly gene, Streptoccocuspneumoniae, ply gene, anthrax, several genes, etc.); b) once theconditions are optimized, genomic DNA (5-6 Mbp) will be tested targetingcharacteristic gene sequences; c) a complete assay will be tested usinggenomic DNA in Task 4.

Fundamental impedance studies will be performed using an Autolab EcoChemie potentiostat/frequency response analyzer, Model PGSTAT20 (severalare available at Nanogen). The impedance parameters will be examined inthe following range: sinusoidal AC signal at 10-50 mV amplitude;frequency range 20 kHz to 5 Hz between the array electrodes. DCpotential (E_(DC)) will be controlled with respect to Ag/AgO QREsurrounding the electrode array and/or versus an external standardcalomel electrode. The buffer electrolytes will include our standardbuffers for the performance of electronic microarray DNA analysisincluding histidine (concentration 10-100 mM), low salt-buffer(phosphate buffer), and a high salt buffer (phosphate buffer with sodiumchloride ions). We have developed fixtures which can provide contacts tothe cartridge and the chip. The 400-site miniature prototype systemdeveloped as a part of the DUST program (cf., FIG. 7) which accepts a400-site array/cartridge (cf., FIG. 8) will be used to performelectronic addressing as well as further impedance measurements. Thesystem has a built in fluorescence detection system which will be usedas a verification of the hybridization of particles to the DNA target(fluorescence labeled particles will be used for this purpose) as wellas a DNA attachment to the oligonucleotide probes in the permeationlayer. The CMOS chip has an array of 16×25 (400-sites); each electrodebeing 50 μm in diameter with a 150-μm center-to-center distance. TheCMOS chip is a flip-chip bonded onto a ceramic substrate (0.015″ thick),which is further bonded with a machined cover plate (acrylic) bypressure sensitive adhesive into a cartridge (cf., FIG. 8). Assuming thelength of DNA at ca 0.34 nm per base pair, DNA bridging experiments willuse 20-40,000 bp DNA templates. The size of these DNAs is therefore inthe range between 2-4 microns and they will be capable of bridging theelectrode distance of the newly designed chip.

FIG. 7. Nanogen's portable prototype instrument with the electroactivemicro-array and optical detection. The instrument is operated by alaptop (left). Components of the instrument include the cartridge inletport, reagent reservoirs, peristaltic pumps, electronic control andoptical detection system with a ccd camera (right).

FIG. 8. Photograph of the 400-site CMOS ACV400-chip cartridge and array.Four counter-electrodes, two longitudinally and two horizontallypositioned surround the active working electrode array.

Design and Fabrication of an Electronic Microarray and Cartridge withthe Electrode Array Geometry Suitable for the Proposed Detection System

The main goal of this task is to develop a microelectronic array withthe electrode geometry that will assure highest AC signals for theproposed detection mechanism. This may involve decreasing the spacingand diameter of the electrodes. It is envisioned that the diameter ofthe electrodes in the range between 3-5 microns with approximatelysimilar spacing will provide a higher AC signal, in particular it willincrease chances to achieve the bridging between the two electrodesites. This level of line resolution can be achieved using the samelithographic techniques used in the production of the current 400-sitearray (RF sputtering for platinum deposition and plasma enhancedchemical vapor deposition techniques (PECVD) for insulating silicondioxide deposition). All the equipment is available and methodsestablished at Nanogen for a production of such chip. The maskfabrication and flip-chip processes will be performed using standardvendors. The hydrogel permeation layer is fabricated in-house usingautomated micro-molding and UV curing equipment. The experiments in thistask will involve the use of larger oligonucleotide probes for bridgingthe gap (e.g., 1,000-10,000 based pairs) between the neighboringelectrode array sites as well as smaller nucleotides probes hybridizedto those probes having sequences specific to the template DNA.

Demonstration of Amplification-Less, Rapid and Sensitive Detection ofDNA Target Molecules

This task will focus on defining and testing other important parametersfor the optimization of the AC impedance characterization of the DNAtarget metallization. The DNA targets will be relatively shortoligonucleotides mimicking the PCR amplified DNA samples. Their lengthwill range between few tens to few hundred base pairs. The shortertemplates will be obtained using PCR amplification and purification ofthe product. We have a number of DNA templates which are used as wellcontrolled DNA samples such as Factor II or Factor V sequences. Theoligonucleotide probes will range between 50-80 base pairs to assure ahigh specificity for the DNA template. The experiments planned in thistasks will involve optimization of the metallic particles tags withrespect to: a) particle diameter—ranging from 10 nm to 1 μm diameter; b)charge of particles: carboxylated particles with positive and aminatedparticles with the negative charge will be used in the cyclicelectrophoretic measurements with electrode polarity reversal to enhancethe AC signal as explained earlier; c) oligonucleotide labels: one setof particle tags will be labeled with both sequences complementary tothe DNA target as well as with poly-T (or similar repetitive sequence)for reporting and hybridization to other particle tags in subsequentelectrophoretic addressing (amplification) cycles; other particle tags(subsequent in addition to the first set) will contain poly-A oligolabels with the sequence complementary to poly-T (or similarcomplementary sequence); d) concentration of oligonucleotide probescoverage on the particle tags—it is envisioned that there will be anoptimum of oligo probes concentration on the particles with respect toachieving an efficient clustering of particle tags on the DNA template;e) the number of particle tags with oligonucleotide probes complementaryto the DNA target template—the number of complementary tags alignedalong the DNA will be optimized with respect to the size of DNA targetand size of particles to achieve conditions for fastest seeding and mostefficient metallization.

Several binding techniques between the particle and oligo probes may betested; however, commercially available particles will be used wheneverpossible. DNA oligonucleotide probes can be covalently coupled directlyto the beads to get a surface coverage ranging from only few probes toas large as 10⁵ per particle. The oligonucleotide particle tags couldconsist of poly dT, poly dA, poly dC or poly dG sequences. In addition,specific capture sequences can be added to these beads or a mixture ofbeads may be used. Alternately the beads can be covalently modified withstreptavidin and then used to bind biotinylated oligonucleotide probes.Carboxyl and amine terminated particles, and amine terminated quantumdots are available from several vendors including Polysciences, MoSci,Nanosphere, Pierce, Seradyn, Dynal and Quantum Dot. Several reagents canbe used to covalently couple streptavidin or DNA directly to the beads.For positively charged, aminated beads glutaraldehyde can be used toactivate the beads followed by the addition of 5′ or 3′-amino modifiedDNA sequences at the right concentration to achieve a controlled densityof probes per bead. Alternatively, streptavidin can be added to theglutaraldehyde activated beads and covalently coupled through theterminal lysine residues on the streptavidin subunits. The linked beadsare then passivated using monoethylamine to maintain a net positivesurface charge and to react with the remaining aldehyde linkages.

For negatively charged, carboxylated, beads1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) can be used toactivate the beads followed by the addition of 5′ or 3′-amino modifiedDNA sequences at the appropriate dilution to achieve a controlleddensity of probes per bead. Alternatively streptavidin can be added tothe EDC activated beads and covalently coupled through the terminallysine residues on the streptavidin subunits. The linked beads are thenpassivated using glycine to maintain a net negative surface charge andto react with the remaining o-acylisourea linkages.

Large size DNA templates will be used to test the probability andconditions in DNA template/oligonucleotide probe bridging experiments.Those may include cloned plasmids in the size range from 5 to 20 kb basepairs available commercially (e.g., Invitrogen offers lyophilizedplasmids in variety of sizes, e.g., pREP4, an episomal mammalianexpression vector, Catalog #V004-50, has 10.3 kb. A series ofrestriction enzymes are provided which can be used to cut the plasmid toa desired length (e.g., Aatl will provide only one cut on pREP4). Theplasmids with known sequences will be used which will simplify thedesign of the oligonucleotide probes for sensor applications. Theselonger probes will be attached to neighboring electrodes to providelonger arms for bridging with the DNA template and extended particlestags. Once metallized (as described earlier), the current will flow overthe metallized bridge and provide an extremely high impedancesignal-to-noise ratio because a short will be created between the twoelectrodes. It is noteworthy that the bridging between the two electrodesites could be made of several pieces of single stranded DNA attached toeach other at their ends or through metallic particle tags, thusproviding a longer stretch between the electrodes.

Design and Testing of a Representative Assay and Validation of theDetection System

To properly validate the proposed detection method DNA target sampleswith accurately known sequence will be used in the assay design. We haveseveral relevant plasmid constructs as well as genomic DNAs with knownsequences, e.g., vaccinia plasmid, or pla plasmid (plague) which wereobtained from USAMIID. This task will examine aspects of performing anentire assay including the potential for integration with the samplepreparation steps. The portable instrument developed through the DUSTprogram could accommodate both sample preparation and proposed newdetection system.

It is envisioned that the antibody or oligonucleotide labeled magneticparticles could be used in the proposed detection technique. This willenable integration with simple sample preparation steps which willconsist of magnetic separation of pathogens from the sample usingantibody labeled beads (through the DUST program we have developed anumber of antibody labeled beads specific for several infectious diseasepathogens, e.g. E. Coli, S. typhimurium, S. pneumoniae, etc). Thepathogens (or cells of interest) are thus first separated magneticallyand subjected to lysis (we have demonstrated that simple thermal lysissteps were satisfactory to efficiently separate and confirm pathogenlevels as low as 10-100 per ml). If necessary, released genomic DNAcould be first enzymatically cut to a precise number of cuts with knownsequences. The DNA target is captured on the microarray by electronicaddressing to the biotinilated oligonucleotide probes on the hydrogelpermeation layer. The oligonucleotides labeled metallic particles arethen added and the assay performed as described earlier for the ACimpedance detection of DNA target metallization. This task will resultin the optimization of the assay steps and will evaluate ruggedness andreproducibility as well as the sensitivity of the proposed electronicdetection method. The validation performed for the PCR amplifiedsequences of interest, DNA size 200-1,000 bp as well as for therepresentative genomic DNA (4-6 Mbp, cut in pieces enzymatically or bythermal treatment in the sample preparation process).

It will be apparent to those skilled in the art that modifications maybe made without departing from the spirit and scope of the invention.Accordingly, it is not intended that the invention be limited except asmay be necessary in view of the appended claims.

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1. A method for the electronic detection of hybridized targetscomprising the steps of: providing an electronic microarray havingspecific probes associated with two or more microarray locations,electronically accumulate the target on a particular electrode site atthe micro-electrode array, sequential electronic hybridization ofoligonucleotide labeled conductive particles on the target, andmonitoring the electrochemical AC impedance changes at the electrodesite.
 2. The method of claim 1 wherein the target is a genomic target.3. The method of claim 2 wherein the genomic target is a nucleic acid.4. The method of claim 3 wherein the nucleic acid is DNA.
 5. The methodof claim 3 wherein the nucleic acid is RNA.
 6. The method of claim 1wherein the particles are nanoparticles.
 7. The method of claim 1further including the step of electroplating over the DNA target.
 8. Themethod of claim 4 wherein the target serves as the metallizationtemplate.
 9. The method of claim 1 wherein the conductive particles aremetallic particles.
 10. The method of claim 9 wherein the metallicparticles are gold.
 11. The method of claim 9 wherein the metallicparticles are silver.
 12. The method of claim 9 wherein the metallicparticles are palladium.